Investigation of the effects of overexpression of jumping translocation breakpoint (JTB) protein in MCF7 cells for potential use as a biomarker in breast cancer

Abstract

Jumping translocation breakpoint (JTB) gene acts as a tumor suppressor or an oncogene in different malignancies, including breast cancer (BC), where it was reported as overexpressed. However, the molecular functions, biological processes and underlying mechanisms through which JTB protein causes increased cell growth, proliferation and invasion is still not fully deciphered. Our goal is to identify the functions of JTB protein by cellular proteomics approaches. MCF7 breast cancer cells were transfected with sense orientation of hJTB cDNA in HA, His and FLAG tagged CMV expression vector to overexpress hJTB and the expression levels were confirmed by Western blotting (WB). Proteins extracted from transfected cells were separated by SDS-PAGE and the in-gel digested peptides were analyzed by nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). By comparing the proteome of cells with upregulated conditions of JTB vs control and identifying the protein dysregulation patterns, we aim to understand the function of this protein and its contribution to tumorigenesis. Gene Set Enrichment Analysis (GSEA) algorithm was performed to investigate the biological processes and pathways that are associated with the JTB protein upregulation. The results demonstrated four significantly enriched gene sets from the following significantly upregulated pathways: mitotic spindle assembly, estrogen response late, epithelial-to-mesenchymal transition (EMT) and estrogen response early. JTB protein itself is involved in mitotic spindle pathway by its role in cell division/cytokinesis, and within estrogen response early and late pathways, contributing to discrimination between luminal and mesenchymal breast cancer. Thus, the overexpressed JTB condition was significantly associated with an increased expression of ACTNs, FLNA, FLNB, EZR, MYOF, COL3A1, COL11A1, HSPA1A, HSP90A, WDR, EPPK1, FASN and FOXA1 proteins related to deregulation of cytoskeletal organization and biogenesis, mitotic spindle organization, ECM remodeling, cellular response to estrogen, proliferation, migration, metastasis, increased lipid biogenesis, endocrine therapy resistance, antiapoptosis and discrimination between different breast cancer subtypes. Other upregulated proteins for overexpressed JTB condition are involved in multiple cellular functions and pathways that become dysregulated, such as tumor microenvironment (TME) acidification, the transmembrane transport pathways, glycolytic flux, iron metabolism and oxidative stress, metabolic reprogramming, nucleocytosolic mRNA transport, transcriptional activation, chromatin remodeling, modulation of cell death pathways, stress responsive pathways, and cancer drug resistance. The downregulated proteins for overexpressed JTB condition are involved in adaptive communication between external and internal environment of cells and maintenance between pro-apoptotic and anti-apoptotic signaling pathways, vesicle trafficking and secretion, DNA lesions repair and suppression of genes involved in tumor progression, proteostasis, redox state regulation, biosynthesis of macromolecules, lipolytic pathway, carbohydrate metabolism, dysregulation of ubiquitin-mediated degradation system, cancer cell immune escape, cell-to-cell and cell-to-ECM interactions, and cytoskeletal behaviour. There were no significantly enriched downregulated pathways.

Keywords: Breast cancer; JTB overexpressed condition; jumping translocation breakpoint (JTB) protein; proteomics.

Figure 1

hJTB plasmid with 6X His tag at the N-terminus and FLAG, HA and eGFP tag at its C-terminus for the overexpression with Neomycin and Ampicillin resistance genes customized by Genscript^®.

Figure 2

Workflow for cellular proteomics from 1D-SDS PAGE and in gel-trypsin digestion.

Figure 3

Confocal microscope images showing confirmation of stable transfection for control (A) and overexpressed (B) JTB condition. Left panel is the BF mode, middle panel is the GFP mode and the right panel is a merge between the BF and GFP modes.

Figure 4

Overexpression confirmation of hJTB compared to control samples with (A) showing the overexpression at ~45 kDa in upregulated MCF7 cell lysate compared to control using the commercially available full length hJTB antibody from Invitrogen. (B) shows GAPDH used as the loading control at 37 kDa.

Figure 5

12% SDS-PAGE gel with 200 µg protein from Control and Up (overexpressed hJTB) from MCF7 cell lysates cut into individual gel bands from each lane.